EP3774926A1 - Display systems for proteins of interest - Google Patents
Display systems for proteins of interestInfo
- Publication number
- EP3774926A1 EP3774926A1 EP19726131.6A EP19726131A EP3774926A1 EP 3774926 A1 EP3774926 A1 EP 3774926A1 EP 19726131 A EP19726131 A EP 19726131A EP 3774926 A1 EP3774926 A1 EP 3774926A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- sequence
- seq
- nos
- poi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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Definitions
- Phage display is the oldest and most commonly used selection method. It is based on the presentation of the protein of interest (POI) on the surface of phages which contain the corresponding DNA of the POI, for example, an antibody.
- POI protein of interest
- the POI is covalently linked to a phage coat protein. Most frequently, a genetic fusion to the gene III protein (pill) of filamentous phage Ml 3 is used but fusions to the other coat proteins have been in the literature as well.
- the POI-pIII fusion is usually cloned into a phagemid vector which contains an antibiotic resistance marker, origins of replication for phage and bacterial DNA polymerases, and a phage morphogenetic signal for the packaging of the phagemid into the phage (Qi e/ a/., 2012).
- the missing genes for phage production are supplied by infection with a helper phage, a full-length phage with a mutated packaging signal. Due to this mutation, predominantly the phagemid, and not the helper phage DNA, is packaged into the phage coat. These phages typically contain only one POI-pIII fusion on the surface since the wild-type pill from the helper phage is incorporated faster than the fusion protein from the phagemid. The resulting monovalent protein display allows the selection for protein binding strength as it is not influenced by any avidity effects.
- Multivalent display can be achieved by using hyperphage, a modified helper phage which does not contain a wild-type genelll (gill), therefore, all 5 copies of pill have to originate from the phagemid (Rondot et al ., 2001).
- CysDisplay® the pill on the phagemid carries a cysteine at the N-terminus, and the POI has an additional cysteine at the C -terminus of the heavy chain (Rothe et al ., 2008). Upon export to the periplasm, they form a disulfide bond and are incorporated into the phage coat.
- the advantage of CysDisplay® is the simple and quantitative elution of the specific phage POIs by cleavage of the disulfide bond with a reducing agent like DTT, a method that is independent of POI affinity.
- Non-covalent ways to display a POI on the pill protein are the use of two dimerization domains (Wang et al, 2010) or the protein A ZZ domain (Mazor et al, 2010).
- the POI are usually expressed without the pill most commonly by subcloning into an expression vector, by removing the gene III from the vector by restriction digest and re-ligation or by using an amber stop codon between the POI and the gill in combination with E.coli strains lacking suppressor tRNAs.
- the POI is usually subcloned into an expression vector which gives higher yields than the amber stop codon system. This subcloning step is a laborious and time consuming step especially for high throughput selections against a multitude of targets in parallel.
- sortase system Schomohl et al. , 2014
- a short peptide the sorting motif
- two glycine residues are genetically fused to the N-terminus of a second peptide (or vice versa).
- the sortase enzyme the two modified polypeptides are fused together.
- a disadvantage of this system is the slow enzymatic reaction and the relatively low yield of end product that can be currently achieved.
- Other enzymatic protein ligase systems are butelase (Nguyen et al. , 2014) or peptiligase (Toplak et al, 2016).
- Another example is the in-frame addition of nucleotides encoding one or more cysteines to the C- or N termini of two polypeptides.
- cysteine containing polypeptides When such free cysteine containing polypeptides are mixed under oxidizing conditions, they will form disulfide bridges.
- Such systems suffer from the many side-products that will appear.
- a third example is the so-called Spy Tag/Spy Catcher (Reddington et al. , 2015) system.
- the concept of spontaneous isopeptide formation in naturally occurring proteins has been used to covalently attach one polypeptide to another.
- a domain from the Streptococcus pyogenes protein FbaB that contains such an isopeptide bond has been split into two parts.
- One part, the SpyTag is a 13 amino acid peptide that contains part of the autocatalytic center.
- the other part, the SpyCatcher is a 116 amino acid protein domain containing the other part of the center.
- SpyTag and K-tag are both short peptides that are covalently fused by addition of SpyLigase.
- FIGs. 1A-1D provide an illustration of the phage and cell display (FIGs. 1A-1B) and the disclosed invention (FIGs. 1C-1D).
- FIG. 2 provides an exemplary SpyCatcher-pIII sequence (SEQ ID NOs: 29 and 30).
- FIGs. 3A-3B illustrate Western blot analysis of SpyCatcher phage compared to VCSM13 phage.
- FIG. 3A shows detection with anti-SpyCatcher antibody which can only detect the modified SpyCatcher phage but not the VCSM13 phage.
- FIG 3b shows a Western blot with anti-pill detection which gives a signal for both phages but shows a larger product (SpyCatcher-pIII fusion) for the SpyCatcher phages
- FIG. 4 is a Western blot of Fab phages.
- the Fab expressing phages were analyzed by Western blot with an anti-pill antibody.
- Two main bands are visible, the Fab-SpyTag- SpyCatcher-pIII and SpyCatcher-pIII fusion protein (Figs. 3A-3B).
- the intensity ratio of the two bands indicates an average display rate of approximately 2 Fab per phage (each phage carries ⁇ 5 pill proteins at the tip of the coat).
- FIG. 5 demonstrates proper folding of the Fab on the surface of the phage.
- Fab displaying phages were analyzed by ELISA for antigen binding capability (TNFa for Adalimumab and ErbB2 for Trastuzumab). Both Fab phages demonstrated a strong signal over background value which confirms the functionality of the Fab on the surface of the phage.
- the invention described herein is a protein display selection method which uncouples the POI library from the display selection system. This is achieved by removing the fusion partner (e.g. gllll protein in the case of phage display) from the library vector.
- Display of the POI can be achieved by forming a covalent bond between the POI and the anchor protein post expression either by enzymatic protein ligation (e.g. SpyLigase, SnoopLigase, sortase, butelase, peptiligase etc.) or by spontaneous covalent bond formation (e.g. SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, etc.).
- the POI library is fused to a tethering sequence, for example SpyTag, at the C-terminus of the POI which then forms a covalent bond to a capture sequence found on an anchor protein, for example, the SpyCatcher-fused anchor protein, e.g., a SpyCatcher-genelll protein (SpyCatcher-pIII) fusion, for the most common form of phage display.
- an anchor protein for example, the SpyCatcher-fused anchor protein, e.g., a SpyCatcher-genelll protein (SpyCatcher-pIII) fusion, for the most common form of phage display.
- the POI is fused to the capture protein and the anchor protein is fused to the tether sequence.
- FIG. 1 provides a schematic representation of the disclosed invention.
- the capture-anchor protein gene is not encoded on the library phagemid but either by a modified helper phage, modified E.coli genome or an additional plasmid - for phage display or bacterial display - or by a modified expression host or additional plasmid for other display techniques.
- the display system of this invention expresses the POI for the selection steps without modification i.e. without fusion to an anchor protein. Therefore, the expressed POI during selection is identical to the protein used for the following screening and production steps. Circumventing an artificial fusion (e.g., POI-pIII), which is required in conventional display selection systems, can avoid a bias in the selection introduced by different properties (expression, solubility, activity, stability, etc.) of a POI-anchor fusion protein. Furthermore, since the POI is not expressed as an anchor fusion protein, no subcloning step is required prior to screening and expression. This saves time and effort especially in high throughput selection settings.
- an artificial fusion e.g., POI-pIII
- the library becomes usable for various display selection systems i.e. different coat proteins for phage display (coat proteins other than pill) as well as other display systems (e.g., yeast display, mammalian or bacterial display) as long as the vector elements are compatible with the other host.
- display selection systems i.e. different coat proteins for phage display (coat proteins other than pill)
- other display systems e.g., yeast display, mammalian or bacterial display
- the display system can also be switched within a selection between different selection rounds or after selection for screening e.g. of POI displayed on other hosts.
- a high display rate of the POI on the phage is often beneficial in the first selection round.
- this can be achieved by using hyperphage without a gill leaving the POI-pIII as the only source for pill and thus producing phage with several POI on the phage surface.
- a high display rate can be achieved by infecting E.coli containing the POI library with a modified helper phage which carries a SpyCatcher-gHI instead of gill. All copies of the pill in the phage coat thus carry a SpyCatcher which enables protein ligation of several POI per phage.
- the selected phages of the first round are used to infect a modified E.coli strain which carries a SpyCatcher-gHI fusion in their genome or carries an additional plasmid for co-expression of SpyCatcher-glll.
- the SpyCatcher can be fused to a complete gill (Nl-N2-Ct) or to truncated or modified gill (N2-Ct or Ct).
- bacteria After infection with helper phage, bacteria produce new phages which predominantly carry the wildtype pill from the helper phage as it gets incorporated into the phage coat faster than the modified pill from the E.coli genome/second plasmid.
- an additional SpyTag or a SpyTag fusion protein can be co-expressed which competes with the POI-SpyTag fusion for ligation with SpyCatcher which is introduced by the modified SpyCatcher-pIII helper phage.
- SpyCatcher which is introduced by the modified SpyCatcher-pIII helper phage.
- gill other phage coat proteins can be used.
- non-modified E.coli which do not carry a SpyCatcher- fusion gene are infected with the phage output and can be used for screening and expression.
- the SpyTag at the C-terminus of the POI can be used for detection (with appropriate antibodies against the tag) as well as for conjugation with biotinylated, fluorescent- or enzyme-labelled SpyCatcher.
- one embodiment of the envisioned invention consists of the POI-SpyTag fusion protein and the SpyCatcher-phage coat protein fusion, either produced by a modified E.coli strain, by a modified helper phage or co expressed by a second plasmid to display POI on the surface of phage.
- a POI-tethering sequence fusion protein is linked to a capture sequence-anchor fusion protein to display the POI on the surface of a biological entity.
- the capture sequence-anchor protein fusion is part of, or associated with, the outer surface of the biological entity and thus links the POI to the surface of the biological entity by formation of a covalent bond between the tethering sequence and the capture sequence.
- the biological entity contains the DNA which encodes the POI and thus provides a physical link of the POI and its DNA which is required for protein selection systems.
- the biological entity can be a phage with the capture sequence fused to a phage coat protein such as pill-, pVI-, pVII-, pVIII- or pIX protein or an optimized, modified or truncated version of these coat proteins and the phagemid encoding the POI inside the phage coat.
- the biological entity can be a yeast e.g., Saccharomyces cerevisiae or Pichia pastoris with the capture sequence fused to, e.g., the a-agglutinin yeast adhesion receptor, a bacteria or a mammalian cell.
- the POI can be a protein or a library of proteins such as antibodies, antibody fragments, single chain antibodies (e.g., scFv, scFab), single domain antibodies, protein scaffolds (e.g., based on fibronectin III, cy statin, lipocalins, Ankyrin repeat domains, Z domain of protein A and others) etc.
- the system can be used to select for proteins with certain properties such as affinity, activity or certain biological or physical properties. Depending on the biological entity the selection system would be referred to as phage display, yeast display, bacterial display or mammalian display.
- the POI library is fused to the capture protein and the anchor protein is fused to the tether sequence.
- the system described in the first embodiment uses a SpyTag/SpyCatcher system to spontaneously form a covalent bond and link the POI to the anchor protein.
- the SpyTag or a modified or optimized version of it e.g., SpyTag002
- the tether sequence e.g., the SpyCatcher or a modified or optimized version of it, e.g., SpyCatcher002
- the POI is either fused to the SpyTag and the anchor protein is fused to the SpyCatcher or the POI is fused to the SpyCatcher and the SpyTag is fused to the anchor protein.
- the system described in the first embodiment uses a different protein ligation system than the SpyTag/SpyCatcher to link the POI to the anchor protein such as the SnoopTag/SnoopCatcher (Veggiani et al ., 2016), SpyTag/KTag/SpyLigase (Fierer et al , 2014), Sortase system (Schmohl et al., 2014), split inteins (Thiel et al., 2014), Butelase 1 (Nguyen et al, 2014), Peptiligase (Toplak et al, 2016), or other similar methods.
- SnoopTag/SnoopCatcher Veggiani et al ., 2016
- SpyTag/KTag/SpyLigase Fierer et al , 2014
- Sortase system Schomohl et al., 2014
- split inteins Thiel et al., 2014
- Butelase 1 Ngu
- a POI such as an antibody fragment or single chain antibody library with a tethering sequence at the C-terminus of the heavy or light chain and one or more additional tags which can be used for antibody purification or detection is expressed.
- a filamentous phage such as Ml 3, is the biological entity. Production of phage with the POI displayed on the surface of the phage is achieved by infection of bacteria carrying a phagemid with the POI-tethering sequence fusion gene with a modified helper phage. The modified helper phage produces a capture sequence-pill fusion instead of wildtype pill.
- the modified pill might contain a protease cleavage site between the capture sequence and pill, such as TEV, rhinovirus 3C protease or trypsin, for elution of selected phage by protease cleavage.
- bacteria carrying a plasmid which encodes a capture sequence-pill fusion protein or modified bacteria which carry a capture sequence-pill gene in their genome can be used in combination with helper phage infection to produce new POI displaying phage.
- an optimized, modified or truncated pill fusion might be used.
- a POI such as an antibody fragment or single chain antibody library with a capture sequence at the C-terminus of the heavy or light chain and one or more additional tags which can be used for antibody purification or detection is expressed.
- a filamentous phage such as M13, is the biological entity. Production of phage with the POI displayed on the surface of the phage is achieved by infection of bacteria carrying a phagemid with the POI-capture sequence fusion gene with a modified helper phage. The modified helper phage produces a tethering sequence-pill fusion instead of wildtype pill.
- the modified pill might contain a protease cleavage site between the tethering sequence and pill, such as TEV, rhinovirus 3C protease or trypsin, for elution of selected phage by protease cleavage.
- bacteria carrying a plasmid which encodes a tethering sequence-pill fusion protein or modified bacteria which carry a tethering sequence-pill gene in their genome can be used in combination with helper phage infection to produce new POI displaying phage.
- an optimized, modified or truncated pill fusion might be used.
- the term“genetic constructs” refers to polynucleotide sequences encoding a protein of interest (POI) that is fused, in frame, to a“tether sequence” (TS) or to a capture sequence (CS) or to a protein (also referred to as an“anchor protein”) expressed on the surface or linked to the surface of a prokaryotic cell, eukaryotic cell, prokaryotic virus or phage, or eukaryotic virus that is fused, in frame, to a capture sequence or tether sequence.
- POI protein of interest
- CS capture sequence
- an“anchor protein” protein expressed on the surface or linked to the surface of a prokaryotic cell, eukaryotic cell, prokaryotic virus or phage, or eukaryotic virus that is fused, in frame, to a capture sequence or tether sequence.
- GC1 refers to genetic constructs comprising POI fused to a tether sequence or capture sequence
- GC2 refer
- tether sequence or“tethering sequence” relate to a sequence that is attached to a POI or anchor protein and that facilitates the formation of a covalent linkage to a capture moiety expressed either on the surface of a biological entity or fused to the POI.
- tethering sequences include SpyTag sequences, including SpyTag002, SnoopTag sequences, Sortase motifs, a C-peptide, butelase substrates, and peptiligase substrates.
- the tether sequence may be fused to POI or anchor protein at the N- or C-terminus of such proteins or polypeptides or in an internal loop.
- a spacer sequence may flank the tether sequence in order to enhance accessibility for reaction.
- the spacer may further include a site for specific proteolysis (e.g., by Factor X, thrombin, enterokinase, tobacco etch virus (TEV) NIa protease, rhinovirus 3C protease or trypsin), allowing specific release from a tether sequence.
- a site for specific proteolysis e.g., by Factor X, thrombin, enterokinase, tobacco etch virus (TEV) NIa protease, rhinovirus 3C protease or trypsin
- capture sequence refers to a polypeptide fused to an anchor protein or POI with which a tethering sequence forms a covalent peptide linkage, for example SpyCatcher for the SpyTag tethering sequence.
- the capture sequence may be fused to the anchor protein or POI at the N- or C-terminus of such proteins or polypeptides or in an internal loop.
- a spacer sequence e.g., a glycine/serine rich spacer
- the spacer may further include a site for specific proteolysis (e.g., by Factor X, thrombin, enterokinase, tobacco etch virus NIa protease, rhinovirus 3C protease or trypsin), allowing specific release from a capture sequence.
- a site for specific proteolysis e.g., by Factor X, thrombin, enterokinase, tobacco etch virus NIa protease, rhinovirus 3C protease or trypsin
- prokaryotic system refers to prokaryotic cells such as bacterial cells or prokaryotic viruses, prokaryotic phages or bacterial spores.
- eukaryotic system refers to eukaryotic cells including cells of animal, plants, fungi and protists, and eukaryotic viruses such as retrovirus, adenovirus, baculovirus. Prokaryotic and eukaryotic systems may be, collectively, referred to as“expression systems”.
- expression cassette is used here to refer to a functional unit that is built in a vector for the purpose of expressing recombinant proteins/peptides.
- An expression cassette includes a promoter or promoters, a transcription terminator sequence, a ribosome binding site or ribosome binding sites, and the cDNA encoding a tether sequence or a capture sequence.
- Other genetic components can be added to an expression cassette, depending on the expression system (e.g., enhancers and polyadenylation signals for eukaryotic expression systems).
- vector refers to a nucleic acid molecule, preferably self- replicating, which transfers an inserted nucleic acid molecule into and/or between host cells.
- vectors are circular DNA comprising a replication origin, a selection marker, and/or viral package signal, and other regulatory elements.
- Vector, vector DNA, plasmid DNA, phagmid DNA are interchangeable terms in description of this invention.
- the term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions.
- expression vector is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s).
- expression vector refers to vectors that direct the soluble expression of proteins of interest fused in frame with a tether sequence which is characterized by an ability to form a peptide linkage to capture sequence with a capture sequence produced by a helper vector as disclosed herein.
- the POI may also be fused in frame with a capture sequence which is characterized by an ability to form a peptide linkage to tether sequence with a tether sequence produced by a helper vector as disclosed herein.
- helper vector refers to a genetic system, or host cell-specific vector designed to produce fusion proteins comprising an anchor protein fused in frame with a capture sequence.
- the anchor protein may also be fused in frame with a tether sequence.
- Helper vectors can be introduced into expression systems, in combination with an expression vector, transiently by co-transformation, permanently by integration into host genome, or by viral or phage infection of the host cells.
- display vector set refers to particular combinations of expression vectors (which provide GC1 constructs) and helper vectors (which provide GC2 constructs).
- the co-expression of the GC1 and GC2 result in the display of POI on the surface of an expression system.
- anchor protein refers to a polypeptide or protein of which a portion is found outside the cell membrane or the outer surface of an expression system. Capture sequences or tether sequences are fused to the portion of the anchor protein, (usually the N-terminal portion of the anchor protein) that is found on the outer surface of a cell (see, for example, FIG. 1).
- Non-limiting examples of anchor proteins are provided in Table 1 or have been disclosed in other publications, such as Little et al ., which is hereby incorporated by reference in its entirety.
- captures sequences or tether sequences can be fused, in frame, to coat proteins, such as within genes III, VI, VII, VIII and IX, modified or truncated coat proteins such as long, short or supershort versions of pill.
- capture sequences or tether sequences can be expressed on the surface of bacterial cells, such as E. coli.
- capture sequences can be fused to anchor proteins such as outer membrane proteins (Chang and Lo, 2000; Lee et al., 2004, pili and flagella (Westerlund-Wikstrom et al., 1997), modified lipoproteins (Georgiou et al., 1996), ice nucleation proteins (Jung et al., 1998), or autotransporters (Veiga et al., 2003).
- anchor proteins such as outer membrane proteins (Chang and Lo, 2000; Lee et al., 2004, pili and flagella (Westerlund-Wikstrom et al., 1997), modified lipoproteins (Georgiou et al., 1996), ice nucleation proteins (Jung et al., 1998), or autotransporters (Veiga et al., 2003).
- Capture sequences can also be displayed on the surfaces of eukaryotic host cells, such as Saccharomyces cerevisiae.
- eukaryotic host cells such as Saccharomyces cerevisiae.
- Hwplp, Als3p, Rbt5p in Candida albicans can be used to display capture sequences on yeast cell surfaces.
- mammalian cells can be manipulated to express capture sequences on the cell surface.
- a capture sequence can be expressed on membrane anchor proteins, such as cell surface receptors (Chesnut et al., 1996, J Immunological Methods; Ho et al, 2006, PNAS, 103:9637-9642), GPI anchor sequences (U.S. Patent No. 6,838,446), non- cleavable type 11 signal anchor sequences (U.S. Patent No. 7,125,973).
- polynucleotides As used herein the terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
- polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the nucleotide polymer.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
- polypeptide As used herein the terms “polypeptide”, “peptide”, and “protein,” are used interchangeably herein to refer to polymers of amino acids of any length. POI may, thus, be a polypeptide, peptide or protein.
- A“protein of interest” is any desired polypeptide, peptide, or protein.
- Non- limiting examples of POI include antibodies, for example full length antibodies, antibody fragments, single chain antibodies (e.g., scFv, scFab), or single domain antibodies, protein scaffolds (e.g. based on fibronectin III, cystatin, lipocalins, Ankyrin repeat domains, Z domain of protein A and others), hormones, interleukins, antigens for the development of vaccines, enzymes, etc.
- hGH human growth hormone
- hGH N-methionyl human growth hormone
- bovine growth hormone parathyroid hormone
- thyroxine insulin A-chain
- insulin B-chain proinsulin
- relaxin A-chain relaxin B- chain
- prorelaxin glycoprotein hormones such as follicle stimulating hormones (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH)
- glycoprotein hormone receptors calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, a microbial protein, such as betalactamase
- host cell includes an individual cell or cell culture which can be, or has been, a recipient for the disclosed vectors.
- Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical to the original parent cell due to natural, accidental, or deliberate mutation.
- a polypeptide or polynucleotide sequence is“essentially identical” or “substantially similar” to comparison sequence, if both sequences exhibit substantial amino acid or nucleotide sequence homology.
- essentially identical or substantially identical sequences are at least about 60% identical with each other, after alignment to the homologous regions.
- the sequences are at least about 70% identical, more preferably, they are at least about 80% identical, more preferably, they are at least about 90% identical, of more preferably, the sequences are at least about 95% identical.
- Tether and capture sequences are protein or peptide sequences which can form a covalent bond to each other spontaneously or enzymatically with or without addition or involvement of other factors (proteins, enzymes, catalysts, salts etc.). A few non-limiting examples are listed below.
- U.S. Patent No. 9,547,003 discloses the components of the SpyTag/SpyCatcher system (used as a tether).
- the tether sequence about 5-50 amino acids in length (e.g., about 10, 20, 30, 40 or 50 amino acids in length) and is derived from SEQ ID NOs: 1, 3, 5 or 6.
- Capture sequences are also derived from SEQ ID NOs: 1, 3, 5 or 6 and can be of any length.
- the tether sequence and capture sequence may be fused to POI at the N- or C- terminus of such proteins or polypeptides or in an internal loop.
- a spacer sequence e.g., a glycine/serine rich spacer
- the spacer may further include a site for specific proteolysis (e.g., by Factor X, thrombin, enterokinase or tobacco etch virus NIa protease), allowing specific release from a tether or capture sequence.
- the tether sequence comprises residues 302-308 of the sequence set out in SEQ ID NO: 1, SEQ ID NO: 25 (mgsshhhhhh ssglvprgsv ptivmvdayk rykgsgesgk), SEQ ID NO: 27 (VPTIVMVDAYKRYKS), or a sequence with at least 50% identity to SEQ ID NO: 1, 25 or 27, wherein said peptide tag is less than 50 amino acids in length.
- the tether sequence has at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 1 and is less than 50 amino acids in length.
- the tether sequence may comprise residues 301- 308, 300-308, 299-308, 298-308, 297-308, 296-308, 295-308, 294-308, 293-308, 292-308, 291-308 or 290-308 of SEQ ID NO: 1 or a sequence with at least about 50% to 95% identity to residues 302-308 of SEQ ID NO: 1 or 25.
- the tether sequence comprises the reactive asparagine of position 303 in SEQ ID NO: 1, i.e., this residue is preferably unchanged.
- the tether sequence may be a fragment of SEQ ID NO: 1 or 25 and, in a preferred embodiment, a tether sequence of less than 50 amino acids and which comprises residues 293-308 of the sequence set forth in SEQ ID NO: 1 or which comprises a sequence with at least 50% identity thereto is used.
- the peptide tags are length restricted and comprise less than 50 amino acid residues. Thus the peptide tags do not comprise the sequence of SEQ ID NO: 1 but only specific fragments thereof, or sequences with at least 50% identity e.g., 75, 80, 85, 90 or 95% identity to such specific fragments.
- Other embodiments utilize SEQ ID NO: 25 or a sequence having at least 50% sequence identity thereto as a tether sequence.
- the capture sequence comprises or consists of residues 31-291 of the sequence set out in SEQ ID NO: 1, SEQ ID NO: 26 (msyyhhhhhh dydipttenl yfqgamvttl sglsgeqgps gdmtteedsa thikfskrde dgrelagatm elrdssgkti stwisdghvk dfylypgkyt fvetaapdgy evataitftv neqgqvtvng eatkgdaht), SEQ ID NO: 28 (yfqgamvttl sglsgeqgps gdmtteedsa thikfskrde dgrelagatm elrdssgkti stwisdghvk dfylypgkyt fvetaapdgy evataitftv neqg
- the capture sequence should contain the reactive lysine corresponding to position 179 of SEQ ID NO: 1.
- the binding partner comprises residues 31-292, 31-293, 31-294, 31-295, 31-296, 31-297, 31-298, 31-299, 31-300, 31-301 or 31-302 of the sequence set forth in SEQ ID NO: 1 or a sequence with at least 70% identity thereto, excluding the sequence of SEQ ID NO: 1.
- a tether sequence may be designed from the major pilin protein Spy0l28 using the alternative isopeptide bond in the N-terminus. Therefore, tether sequence may be designed or is obtainable from an N-terminal fragment of the isopeptide protein and the remaining, truncated or overlapping protein fragment may constitute the capture sequence.
- the reactive lysine involved in the isopeptide bond at the N-terminus is found at position 36 of SEQ ID NO: 1 and the reactive asparagine involved in the isopeptide bond is found at position 168 of SEQ ID NO: 1.
- the tether sequence comprises the reactive lysine residue in this instance and the capture sequence comprises the reactive asparagine.
- a tether sequence comprises residues 31-40 of the sequence set out in SEQ ID NO: 1 or a sequence with at least 70% identity thereto and is less than 50 amino acids in length.
- the corresponding capture sequence for the above described tether sequence comprises residues 37-304 of the sequence set out in SEQ ID NO: 1 or has a sequence with at least 70% identity thereto, excluding the sequence of SEQ ID NO: 1.
- the reactive residues in the peptide tag and binding partner are not mutated.
- Another tether sequence comprises residues 179-184 e.g., 173-185 of the sequence set out in SEQ ID NO: 3 or has a sequence with at least 50% identity thereto and is less than 50 amino acids in length.
- the capture sequence comprises residues 191-317 e.g., 186-318 of SEQ ID NO: 3 or a sequence having at least 50% identity thereto, excluding SEQ ID NO: 3.
- a tether sequence or a capture sequence is the full length sequence of SEQ ID NO: 3.
- Another tether sequence comprises fragments of SEQ ID NO: 5 that include the asparagine at position 266 (or sequences having at least 50% identity thereto) and a capture sequence comprising fragments of SEQ ID NO: 5 that have at least 50% sequence identity thereto and which comprises the lysine residue at position 149 but which does not include the asparagine at position 266.
- a capture sequence comprising fragments of SEQ ID NO: 5 that have at least 50% sequence identity thereto and which comprises the lysine residue at position 149 but which does not include the asparagine at position 266.
- Neither the tether sequence nor the capture sequence comprise SEQ ID NO: 5.
- the tether sequence comprises a fragment of SEQ ID NO: 6 that includes the aspartic acid residue at position 101 (or a sequence at least 70% identical thereto) and the capture sequence comprises fragments of SEQ ID NO: 6 that contain the reactive lysine of position 15 (or sequences at least 50% identical thereto). Neither the tether sequence nor the capture sequence comprise SEQ ID NO: 6.
- Another embodiment provides for a tether sequence comprising SEQ ID NO: 25 (mgsshhhhhh ssglvprgsv ptivmvdayk rykgsgesgk), SEQ ID NO: 27
- the tether sequence has at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 25 or 27 and is less than 50 amino acids in length.
- the tether sequence comprises the reactive aspartic acid of position 8 in SEQ ID NO: 27, i.e., this residue is preferably unchanged.
- a capture sequence for either of SEQ ID NO: 25, 27 or a sequence with at least 50% identity to SEQ ID NO: 25 or 27, that is 15 to 40 or 50 amino acids in length and contains an aspartic acid of position 8 in SEQ ID NO: 27, comprises or consists of SEQ ID NO: 26 (msyyhhhhhh dydipttenl yfqgamvttl sglsgeqgps gdmtteedsa thikfskrde dgrelagatm elrdssgkti stwisdghvk dfylypgkyt fvetaapdgy evataitftv neqgqvtvng eatkgdaht) or yfqgamvttl sglsgeqgps gdmtteedsa thikfskrde dgrelagatm elrdssgkti stwisdgh
- POI can be attached to exposed surface proteins using the systems described in WO2016/193746 (which is hereby incorporated by reference in its entirety).
- tether sequences are attached to both the POI and an anchor protein, optionally through linker sequences, such as a glycine/serine rich spacer. These tether sequences are then ligated by a ligase that is also encoded by the host cell.
- the tether sequence can, in some embodiments, have a length between 6-50 amino acids, e.g., 7-45, 8- 40, 9-35, 10-30, 11-25 amino acids in length, e.g.
- the peptide ligase may be between 50-300 amino acids in length, e.g., 60-250, 70-225, 80-200 amino acids in length, e.g., it may comprise or consist of 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 amino acids, providing it meets the definitions set forth for the ligase, below.
- Tether sequences attached to an anchor protein correspond to a capture sequence in this aspect of the invention.
- a pair of tether sequences may be derived from any suitable isopeptide protein.
- tether sequences may be derived from the major pilin protein Spy0l28, which has an amino acid sequence as set out in SEQ ID NO: 7 and is encoded by a nucleotide sequence as set out in SEQ ID NO: 8.
- Two isopeptide bonds are formed in the protein.
- One isopeptide bond is formed between lysine at position 179 in SEQ ID NO: 7 and asparagine at position 303 in SEQ ID NO: 7 (the reactive residues).
- the glutamic acid residue which induces the spontaneous isopeptide bond is found at position 258 in SEQ ID NO: 7.
- a pair of tether sequences developed from an isopeptide protein set forth in SEQ ID NO: 7 will preferably comprise a tether sequence comprising a fragment of the protein comprising the reactive asparagine at position 303 and a tether sequence comprising a fragment of the protein comprising the reactive lysine at position 179.
- a fragment of the protein comprising the glutamic acid residue at position 258 can be provided separately, i.e., as a peptide ligase that forms the isopeptide bond.
- Another isopeptide bond in the major pilin protein Spy0l28 occurs between the lysine residue at position 36 of SEQ ID NO: 7 and the asparagine residue at position 168 of SEQ ID NO: 7.
- the glutamic acid residue which induces isopeptide formation is found at position 117 in SEQ ID NO: 7.
- a pair of tether sequences developed from an isopeptide protein set forth in SEQ ID NO: 7 will preferably comprise a tether sequence comprising a fragment of the protein comprising the reactive lysine residue at position 36 and a tether sequence comprising a fragment of the protein comprising the reactive asparagine at position 168.
- a fragment of the protein comprising the glutamic acid residue at position 117 may be provided separately as a peptide ligase.
- An isopeptide bond occurs between a lysine residue at position 181 of SEQ ID NO: 9 (ACE19, a domain of an adhesin protein from E. faecalis) and an asparagine residue at position 294 of SEQ ID NO: 9.
- the bond is induced by an aspartic acid residue at position 213 in SEQ ID NO: 9.
- a pair of tether sequences developed from isopeptide protein set forth in SEQ ID NO: 9 will preferably comprise a tether sequence comprising a fragment of the protein comprising the reactive asparagine residue at position 294 and a tether sequence comprising a fragment of the protein comprising the reactive lysine residue at position 181.
- a fragment of the protein comprising the aspartic acid residue at position 213 may be provided separately as a peptide ligase.
- the collagen binding domain from S. aureus which has an amino acid sequence set out in SEQ ID NO: 10 can also be used.
- the isopeptide bond occurs between lysine at position 176 of SEQ ID NO: 10 and asparagine at position 308 of SEQ ID NO: 10.
- the aspartic acid residue which induces the isopeptide bond is at position 209 of SEQ ID NO: 10.
- a pair of tether sequences developed from the isopeptide protein set forth in SEQ ID NO: 10 will preferably comprise a tether sequence comprising a fragment of the protein comprising the reactive lysine at position 176 and a tether sequence comprising a fragment of the protein comprising the reactive asparagine at position 308.
- a fragment of the protein comprising the aspartic acid residue at position 209 may be provided separately as a peptide ligase.
- FbaB from Streptococcus pyogenes can also be used to provide tether sequences and comprises a domain, CnaB2, which has an amino acid sequence set out in SEQ ID NO: 11, is encoded by the nucleotide sequence set out in SEQ ID NO: 12.
- the isopeptide bond in the CnaB2 domain forms between a lysine at position 15 of SEQ ID NO: 11 and an aspartic acid residue at position 101 of SEQ ID NO: 11.
- the glutamic acid residue which induces the isopeptide bond is at position 61 of SEQ ID NO: 11.
- a pair of tether sequences developed from the isopeptide protein set forth in SEQ ID NO: 11 will preferably comprise a tether sequence comprising a fragment of the protein comprising the reactive lysine at position 15 and a tether sequence comprising a fragment of the protein comprising the reactive aspartic acid at position 101.
- a fragment of the protein comprising the glutamic acid residue at position 61 may be provided separately as a peptide ligase.
- the RrgA protein is an adhesion protein from Streptococcus pneumoniae, which has an amino acid sequence as set out in SEQ ID NO: 13 and is encoded by a nucleotide sequence as set out in SEQ ID NO: 14.
- An isopeptide bond is formed between lysine at position 742 in SEQ ID NO: 13 and asparagine at position 854 in SEQ ID NO: 13. The bond is induced by a glutamic acid residue at position 803 in SEQ ID NO: 13.
- a pair of tether sequences developed from the isopeptide protein set forth in SEQ ID NO: 13 will preferably comprise a tether sequence comprising a fragment of the protein comprising the reactive asparagine at position 854 and a tether sequence comprising a fragment of the protein comprising the reactive lysine at position 742.
- a fragment of the protein comprising the glutamic acid residue at position 803 may be provided separately as a peptide ligase as defined above.
- the PsCs protein is a fragment of the por secretion system C-terminal sorting domain protein from Streptococcus intermedius, which has an amino acid sequence as set out in SEQ ID NO: 15 and is encoded by a nucleotide sequence as set out in SEQ ID NO: 16.
- An isopeptide bond is formed between lysine at position 405 in SEQ ID NO: 15 and aspartate at position 496 in SEQ ID NO: 15.
- a pair of tether sequences developed from the isopeptide protein set forth in SEQ ID NO: 15 will preferably comprise a tether sequence comprising a fragment of the protein comprising the reactive aspartate at position 496 and a tether sequence comprising a fragment of the protein comprising the reactive lysine at position 405.
- tether sequences may be derived from an isopeptide protein comprising an amino acid sequence as set forth in any one of SEQ ID NO: 21, 23, 25 or 27 or a protein with at least 70% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NO: 21, 23, 25 or 27.
- said isopeptide protein sequence above is at least 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identical to the sequence (SEQ ID NO: 21, 23, 25 or 27) to which it is compared.
- sortase enzymes and sortase recognition and bridging domains.
- Schmohl et al. (2014) which is hereby incorporated by reference in its entirety, discuss sortase mediated ligation for the site- specific modification of proteins.
- the sortase recognition and bridging domains are considered tethering sequences and the tethering sequence fused to an anchor protein corresponds to a capture sequence in this aspect of the invention.
- Sortases are transpeptidases produced by Gram-positive bacteria to anchor cell surface proteins covalently to the cell wall.
- the Staphylococcus aureus sortase A (SrtA) cleaves a short C-terminal recognition motif (LPXTG (SEQ ID NO: 17) (referred to herein as a sortase recognition domain).
- the sortase recognition domain is a sortase A recognition domain or a sortase B recognition domain.
- the sortase recognition domain comprises or consists of the amino acid sequence: LPTGAA (SEQ ID NO: 18), LPTGGG (SEQ ID NO: 19), LPKTGG (SEQ ID NO: 20), LPETG (SEQ ID NO: 21), LPXTG (SEQ ID NO: 22) or LPXTG(X) n (SEQ ID NO: 23), where X is any amino acid, and n is 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, in the range of 0-5 or 0-10, or any integer up to 100.
- the sortase recognition domain can be fused, in frame, to a POI or an anchor protein, optionally through a gly cine/ serine rich spacer. Where it is attached to a POI, it is considered a tether sequence and where it is attached to an anchor protein, it is considered a capture sequence.
- the sortase A bridging domain comprises one or more glycine residues at one of its termini.
- the one or more glycine residues may optionally be: Gly, (Gly) 2 , (Gly) 3 , (Gly) 4 , or (Gly) x , where x is an integer of 1-20.
- the sortase bridging domain can be attached to a POI or an anchor protein, optionally through a glycine/serine rich spacer. Where it is attached to a POI, it is considered a tether sequence and where it is attached to an anchor protein, it is considered a capture sequence.
- the sortase B recognition domain comprises the amino acid sequence NPX1TX2, where XI is glutamine or lysine; X2 is asparagine or glycine; N is asparagine; P is proline and T is threonine.
- the sortase recognition domain can be fused, in frame, to a POI or an anchor protein, optionally through a glycine/serine rich spacer. Where it is attached to a POI, it is considered a tether sequence and where it is attached to an anchor protein, it is considered a capture sequence.
- the sortase B bridging domain comprises one or more glycine residues at one of its termini.
- the one or more glycine residues may optionally be: Gly, (Gly) 2 , (Gly) 3 , (Gly) 4 , or (Gly) x , where x is an integer of 1-20.
- the sortase bridging domain can be attached to a POI or an anchor protein, optionally through a glycine/serine rich spacer. Where it is attached to a POI, it is considered a tether sequence and where it is attached to an anchor protein, it is considered a capture sequence.
- Yet another means for tethering a POI to an anchor protein comprises the use of butelase 1 to form a peptide bond between the butelase recognition motif (where Asx is Asn or Asp) and the amino terminus of another polypeptide.
- the Asx-His-Val motif can be fused, in frame, to a POI or an anchor protein, optionally through a glycine/serine rich spacer.
- Butelase can then be used to form a peptide bond between the Asx-His-Val motif and the N-terminal amino acid of the anchor protein.
- WO 2017/058114 which is hereby incorporated by reference in its entirety, discloses methods and materials for butelase- mediated peptide ligation.
- split inteins can exist as two fragments encoded by two separately transcribed and translated genes. These so-called split inteins self-associate and catalyze protein- splicing activity in trans.
- Split inteins have been identified in diverse cyanobacteria and archaea (Caspi et al., 2003; Choi J. et al., 2006; Dassa B. et al., 2007; Liu X. and Yang L, 2003; Wu H. et al., 1998; and Zettler J. et al., 2009, the disclosures of which are hereby incorporated by reference in their entireties).
- Thiel et al. (2014) and WO 2013/045632 each of which is hereby also incorporated by reference in its entirety, also disclose the use of split inteins that can be used to fuse heterologous proteins.
- the vectors of the present invention generally comprise transcriptional or translational control sequences required for expressing the POI and anchor proteins.
- Suitable transcription or translational control sequences include but are not limited to replication origin, promoter, enhancer, repressor binding regions, transcription initiation sites, ribosome binding sites, translation initiation sites, and termination sites for transcription and translation.
- the origin of replication permits replication of the vector in a suitable host cell.
- the choice of ori will depend on the type of host cells and/or genetic packages that are employed.
- the expression vector typically comprises two ori sequences, one directing autonomous replication of the vector within the prokaryotic cells, and the other ori supports packaging of the phage particles.
- Preferred prokaryotic ori is capable of directing vector replication in bacterial cells. Non-limiting examples of this class of ori include pMBl, pUC, as well as other E. coli origins.
- Preferred ori supporting packaging of the phage particles includes but is not limited to fl ori, Pfi phage replication ori.
- eukaryotic origins of DNA replication In the eukaryotic system, higher eukaryotes contain multiple origins of DNA replication, but the ori sequences are not clearly defined.
- the suitable origins of replication for mammalian vectors are normally from eukaryotic viruses.
- Preferred eukaryotic ori include, but are not limited to, SV40 ori, EBV ori, or HSV ori.
- a“promoter” is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region located downstream (in the 3' direction) from the promoter. It can be constitutive or inducible. In general, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and“CAT” boxes.
- promoters will largely depend on the host cells in which the vector is introduced.
- a variety of robust promoters are known in the art.
- Preferred promoters are lac promoter, Trc promoter, T7 promoter and pBAD promoter.
- the prokaryotic promoter can be placed immediately after the eukaryotic promoter, or inside an intron sequence downstream of the eukaryotic promoter.
- Suitable promoter sequences for eukaryotic cells include the promoters for 3- phosphoglycerate kinase, or other glycolytic enzymes, such as enolase, glyceraldehyde- 3phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6phosphate isomerase, 3 -phosphogly cerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
- 3- phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde- 3phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6phosphate isomerase, 3 -phosphogly cerate muta
- promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
- Preferred promoters for mammalian cells are SV40 promoter, CMV promoter, b-actin promoter and their hybrids.
- Preferred promoters for yeast cell includes but is not limited to GAL 10, GAL I, TEFI in S. cerevisiae, and GAP, AOX1 in P. pastoris.
- the termination sequences associated with the exogenous sequence are also inserted into the 3' end of the sequence desired to be transcribed to provide polyadenylation of the mRNA and/or transcriptional termination signal.
- the terminator sequence preferably contains one or more transcriptional termination sequences (such as polyadenylation sequences) and may also be lengthened by the inclusion of additional DNA sequence so as to further disrupt transcriptional read-through.
- Preferred terminator sequences (or termination sites) of the present invention have a gene that is followed by a transcription termination sequence, either its own termination sequence or a heterologous termination sequence.
- termination sequences include stop codons coupled to various yeast transcriptional termination sequences or mammalian polyadenylation sequences that are known in the art and are widely available.
- the terminator comprises a gene
- the vectors may contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will survive and/or grow under selective conditions.
- Typical selection genes encode protein(s) that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, kanamycin, neomycin, zeocin, G418, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media.
- the choice of the proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art.
- the expression vector is a shuttle vector, capable of replicating in at least two unrelated host systems.
- the vector generally contains at least two origins of replication, one effective in each host system.
- shuttle vectors are capable of replicating in a eukaryotic host system and a prokaryotic host system. This enables detection of protein expression in the eukaryotic host (the expression cell type) and amplification of the vector in the prokaryotic host (the amplification cell type).
- one origin of replication is derived from SV40 or 2u and one is derived from pUC, although any suitable origin known in the art may be used provided it directs replication of the vector.
- the vector is a shuttle vector, the vector preferably contains at least two selectable markers, one for the expression cell type and one for the amplification cell type. Any selectable marker known in the art or those described herein may be used provided it functions in the expression system being utilized.
- the vectors encompassed by the invention can be obtained using recombinant cloning methods and/or by chemical synthesis.
- a vast number of recombinant cloning techniques such as PCR, restriction endonuclease digestion and ligation are well known in the art, and need not be described in detail herein.
- One of skill in the art can also use the sequence data provided herein or that in the public or proprietary databases to obtain a desired vector by any synthetic means available in the art.
- appropriate sequences can be excised from various DNA sources and integrated in operative relationship with the exogenous sequences to be expressed in accordance with the present invention.
- This example describes the covalent display of human Fab fragments, adalimumab and trastuzumab, with a SpyTag at the C-terminus of the heavy chain on the surface of Ml 3 phages which have a SpyCatcher fused to the N-terminus of the pill.
- SpyTag and SpyCatcher form a covalent bond within the E.coli that leads to a covalent linkage of the Fab to the pill phage coat protein.
- VCSM13 helperphage were modified to replace the gill with a SpyCatcher-gHI genetic fusion. Cloning was done by ligation of restriction digested PCR products. The phage genome was PCR amplified and restriction sites were inserted with primers at the beginning of the gill (forward primer with Xbal site) and before the start codon of the gill (reverse primer with EcoRI site) omitting the gill signal peptide. The inserted sequence with matching restriction sites consists of a DsbA signal peptide, SpyCatcher, TEV protease cleavage site and a short linker (GGGGSGGGGS, FIG. 2).
- XL1F carrying the modified helperphage genome were grown in 50 ml 2xYT with kanamycin at 37°C overnight. Next day, six 300 ml cultures were inoculated from the overnight culture to OD600 of 0.1 and grown in 2xYT with kanamycin at 37°C to an OD600 of ⁇ 0.6 and then transferred to l8°C for helperphage production overnight. Bacteria were spun down and phage were purified and concentrated from the supernatant by PEG precipitation as described below. The phage pellet was dissolved in 1000 m ⁇ PBS and after addition of 20% glycerol aliquots were stored at -80°C.
- XL1 Blue F’, MC1061 F’ or TG1F’ cells were grown in 2xYT to an OD600 of 0.6- 0.8 and then infected with the modified helperphage for 45 min without shaking and 45 min with shaking.
- the culture was incubated on a shaker at 37°C for 3-4 h and was then transferred to a larger volume of 2xYT with 50 pg/ml kanamycin (e.g. 50 ml culture and 350 ml 2xYT) for overnight phage production at l8°C.
- 50 pg/ml kanamycin e.g. 50 ml culture and 350 ml 2xYT
- the next day the phages were precipitated as described below, dissolved in 400 m ⁇ PBS and stored at -80°C after addition of 20% glycerol.
- Phage PEG precipitation Phage were precipitated from the overnight culture supernatant by addition of 1 ⁇ 4 the volume +10% of ice cold PEG/NaCl solution (20% PEG 6000, 2.5M NaCl). Supernatant was incubated on ice on a shaker for 30 min and phages were centrifuged at 13,000 x g for 60 min at 2-8°C. The supernatant was discarded and the pellet resuspended in 10 ml PBS. Remaining bacteria were removed from the phage solution by sterile filtration with a 0.22 pm filter and PEG/NaCl precipitation was repeated as described. The phage pellet was dissolved in PBS.
- Phage titer and infectivity were determined by spot titration and by ELISA.
- spot titration a culture was inoculated with a TG1F’ colony from a M9 minimal agar plate and grown to OD600 of 0.6-0.8 at 37°C.
- a 1 : 10 dilution series of the helperphage in 2xYT was prepared and lOpl of the dilution was mixed with 90 pl TG1F’ culture, incubated at 37°C for 30 min and then 5 pl were plated on a LB agar plate containing 1% glucose and 50 pg/ml kanamycin and incubated at 37°C overnight.
- ETnmodified VCSM13 helperphages were used as a reference. Colonies in spots with less than 30 colonies were counted and the titer was calculated according to the dilution and used volumes. Titers were -10 11 cfu/ml.
- Determination of the phage titer and proof of display of the SpyCatcher on the phage surface was done by ELISA.
- An ELISA microtiter plate was coated at 4°C overnight with an anti-Ml3 antibody (anti-pVIII, 1 pl/ml in PBS) and anti-SpyCatcher Fab (5 pg/ml in PBS), respectively.
- an anti-Ml3 antibody anti-pVIII, 1 pl/ml in PBS
- anti-SpyCatcher Fab 5 pg/ml in PBS
- Detection was performed with an anti -Ml 3 HRP detection antibody and QuantaBlu peroxidase fluorescence substrate.
- the titer estimated by this method was at ⁇ 5xl0 13 phage/ml.
- the ELISA with anti- SpyCatcher capture antibody gave a strong signal thus confirming the presence of phage with one or more SpyCatchers displayed on the surface.
- Presence of the SpyCatcher on the surface of the phage was also confirmed by Western blot of denatured phage with an anti-SpyCatcher antibody and an anti-pill antibody, respectively.
- a Bio-Rad Mini-PROTEANTM Vertical Electrophoresis Cell was used along with a Mini-Protean TGX 4-20% gel and the Bio-Rad Precision Plus Protein Standard molecular weight marker. Proteins were blotted to a PVDF membrane by using the Bio-Rad Trans-Blot Turbo Transfer System. The membrane was blocked with milk in TBST for lh on a shaker followed by incubation for lh with either one of the two primary antibodies. Secondary antibody for the anti-SpyCatcher primary was an HRP conjugated anti-hFab antibody, for anti-pill an HRP conjugated anti-mouse IgG antibody was used. Clarity Western ECL substrate was used for detection.
- the Western blot with anti-SpyCatcher detection shows a band for SpyCatcher phages only but not for VCSM13 phages (negative control; FIG. 3A).
- the blot with anti-pill detection shows that the pill band in SpyCatcher phages runs higher than VCSM13 phages.
- the shift equalt the increase in molecular weight by the SpyCatcher fused to the pill (FIG. 3B).
- Human Fab antibody fragments (trastuzumab and adalimumab) with a SpyTag (Spy) and hexahistidine tag (H) at the C-terminus of the heavy chain were cloned into a bicistronic expression vector pBBxl, consisting of a lac promoter, Shine-Dalgarno (SD) sequence, start codon, pelB leader sequence, Fab light chain stop codon, SD sequence, start codon, ompA leader sequence Fab heavy chain, SpyTag, His tag, and stop codon.
- the vector furthermore carries a ColEl and fl origin of replication, a chloramphenicol resistance and lac repressor (Lacl).
- the plasmids described above were transformed into TG1F’ cells.
- a freshly inoculated culture was grown overnight in 5 ml 2xYT with chloramphenicol and 1% glucose at 37°C on a shaker.
- a 100 ml culture was inoculated with this preculture to an OD600 of 0.05 and grown at 37°C to an OD600 of 0.5.
- 50 ml of this culture were infected with SpyCatcher-pIII helperphages at 37°C for 45 min without shaking and 45 min with shaking. Bacteria were spun down and the supernatant was discarded.
- the pellet was resuspended in 300 ml 2xYT with chloramphenicol, kanamycin, and 0.25 mM IPTG and cultured overnight at 30°C for phage production. Bacteria were spun down and phages were PEG precipitated from the supernatant as described before. The phage pellet was dissolved in 500-1000 m ⁇ PBS and stored at -80°C after addition of 20% glycerol.
- Phage ELISA was performed as described above for the SpyCatcher-pIII phages.
- Fab displaying phages with known titer were used as a standard to determine the phage titer. Titers determined with this method were in the range of lxlO 14 phages/ml.
- an anti-Fd capture antibody was used for the second ELISA.
- This antibody can capture phages which carry a Fab heavy chain (Fd) on the surface of the phages and thus this assay confirms a successful Fab covalent capture display.
- This ELISA gave a strong signal thus indicating a good display rate of most likely several Fab per phage.
- Fab phages were analyzed by Western blot with an anti-pill antibody. Western blot was performed as described above for SpyCatcher-pIII phages. Two main bands are visible, the Fab-SpyTag-SpyCatcher-pIII and SpyCatcher-pIII fusion protein (FIG. 4). The intensity ratio of the two bands indicates an average display rate of approximately 2 Fab per phage (each phage carries ⁇ 5 pill proteins at the tip of the coat).
- Fab displaying phages were also analyzed by ELISA for antigen binding capability.
- Fab antigens, TNF-a for Adalimumab and ErbB2 for Trastuzumab were coated on an ELISA plate at 5 pg/ml in PBS overnight at 4°C. After blocking with Chemiblocker in TBST for 1 h, the plate was incubated with a 1 :500 dilution of the Fab phage in TBST for lh. Phages were detected with an HRP- conjugated anti -Ml 3 antibody and QuantaBlu fluorescence substrate. For both Fab phages a strong signal over background value confirmed functionality of the Fab on the surface of the phage (FIG. 5)
- Phage antibodies filamentous phage displaying antibody variable domains. Nature 348:552-554.
- Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol. 10:732-738.
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